WO2015005632A1 - Dll4와 vegf에 특이적으로 결합하는 신규 이중표적 단백질 및 이의 용도 - Google Patents

Dll4와 vegf에 특이적으로 결합하는 신규 이중표적 단백질 및 이의 용도 Download PDF

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WO2015005632A1
WO2015005632A1 PCT/KR2014/006090 KR2014006090W WO2015005632A1 WO 2015005632 A1 WO2015005632 A1 WO 2015005632A1 KR 2014006090 W KR2014006090 W KR 2014006090W WO 2015005632 A1 WO2015005632 A1 WO 2015005632A1
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dll4
cancer
protein
seq
vegf
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PCT/KR2014/006090
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English (en)
French (fr)
Korean (ko)
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이동헌
문경덕
최유빈
강경재
김동인
안진형
유원규
정진원
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한화케미칼 주식회사
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Priority to ES14823338T priority Critical patent/ES2742855T3/es
Priority to CN201480049434.9A priority patent/CN105518028B/zh
Priority to CA2917402A priority patent/CA2917402C/en
Priority to US14/903,077 priority patent/US10184010B2/en
Priority to AU2014287984A priority patent/AU2014287984B2/en
Priority to EP14823338.0A priority patent/EP3020731B1/en
Priority to RU2016104057A priority patent/RU2648154C2/ru
Priority to JP2016525275A priority patent/JP6283411B2/ja
Priority to PL14823338T priority patent/PL3020731T3/pl
Publication of WO2015005632A1 publication Critical patent/WO2015005632A1/ko

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Definitions

  • the present invention provides a double protein comprising a protein that specifically binds to a novel delta-like ligand 4 (DLL4) and an antibody that specifically binds to Vascular endothelial cell growth factor (VEGF). It relates to a target protein.
  • DLL4 novel delta-like ligand 4
  • VEGF Vascular endothelial cell growth factor
  • Notch signaling has been reported to be evolutionarily well preserved, from vertebrates to invertebrates, and plays an important role in determining cell fate early in development. Notch signaling is known to be a major factor that regulates the differentiation process of nerves, eyes, lymph, muscle, blood cells, etc. It is also involved in the development of blood vessels.
  • Notch 1, 2, 3 and 4 there are four Notch receptors (Notch 1, 2, 3 and 4), each Notch receptor is synthesized as a 300-350 kDa protein, and in the Golgi apparatus, S1 by furin-like convertase. The site is cut to form a heterodimer on the cell surface.
  • five notch ligands Jagged-1 / 2 and Delta-like ligand (DLL) 1/3/4) were identified in mammals.
  • Activated Notch signaling is known to cause tumorigenesis in several cancer models.
  • the expression of NICD, an active notch, in rat hematopoietic cells causes T-cell leukemia / lymphomas and about 50% of activated Notch 1 was found in T-ALL acute lymphoblastic leukemia (T-ALL).
  • T-ALL T-ALL acute lymphoblastic leukemia
  • overexpression of activated Notch 4 receptor was found in mice in which MMTV (mouse mammary tumor virus) was inserted (Czech II) and mammary tumors were reported in these mice.
  • Notch receptors, ligands and targets of Notch signaling are activated in various cancers such as cervical cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer and prostate cancer.
  • Notch 1 receptor expression is associated with poor prognosis in breast cancer patients and is known to be associated with cancer metastasis in prostate cancer.
  • DLL4 Delta Like Ligand 4
  • DLL4 is one of the delta classes of ligands that accept Notch protein overexpressed in vascular endothelial cells and is known as a key factor in regulating angiogenesis. have. DLL4 specifically binds Notch 1 or Notch 4 receptors that are overexpressed in the vascular endothelium. DLL4 is also expressed in normal blood vessels, but is known to be overexpressed in cancer blood vessels. Angiogenesis refers to the mechanism by which new blood vessels are formed from existing blood vessels. In particular, neovascularization in tumors requires VEGF (Vascular endothelial growth factor) to receive oxygen and nutrients in the hypoxia region of cancer tissues.
  • VEGF Vascular endothelial growth factor
  • Angiogenesis is caused by angiogenic factors such as). Angiogenesis in tumors is known to play an important role in metastasis as well as tumor growth. Blocking Notch signaling by DLL4 in tumors can inhibit cancer growth because of poor regulation of angiogenesis. In addition, when inhibiting Notch signaling by DLL4, autoimmune diseases can be treated by increasing the number of regulatory T cells (Treg) (US Patent Publication No. 2011-0189200). For this reason, DLL4 has become a new target in the treatment of cancer and autoimmune diseases.
  • Treg regulatory T cells
  • Avastin (Avastin ® , Genentech / Roche) targeting VEGF is an anti-cancer drug that inhibits neovascularization.
  • VEGF inhibitors transform cancer cells into more aggressive and metastatic cancer cells.
  • DLL4 / Notch signaling pathway One of the new anticancer targets is the DLL4 / Notch signaling pathway.
  • VEGF / VGEFR and DLL4 / Notch signaling pathways affect neovascularization due to different mechanisms of action, and thus, stronger anticancer synergies can be expected when both signaling pathways are simultaneously suppressed. It is expected to be.
  • the present inventors have made intensive efforts to develop dual target proteins that specifically bind to human DLL4 and VEGF to effectively inhibit DLL4 / Notch and VEGF / VEGFR signaling pathways and minimize the risk of immunogenicity.
  • the C-terminal region of the Avastin analog which is an IgG basic form, is linked to a novel ScFv (single-chain variable fragment) form that specifically binds human DLL4.
  • the target protein was newly prepared, and this double-target protein effectively blocks the interaction between VEGF and the VEGF receptor protein, as well as the interaction between DLL4 and Notch protein, thus completing the present invention by showing an excellent anticancer effect. It was.
  • One object of the present invention is a conformational epitope of DLL4 comprising amino acid sequence 58 to 65 and amino acid sequence 110 to 115 in the amino acid sequence of the DLL4 (Delta-like ligand 4) protein represented by SEQ ID NO: 21 It is to provide a dual target protein comprising a protein that specifically binds to DLL4, which recognizes a conformational epitope, and an antibody that specifically binds to Vascular endotelial growth factor (VEGF).
  • VEGF Vascular endotelial growth factor
  • Another object of the present invention is to provide a polynucleotide encoding the dual target protein, an expression vector comprising the polynucleotide, and a transformant comprising the expression vector.
  • Another object of the present invention is to provide a composition comprising the dual target protein.
  • Still another object of the present invention is to provide a pharmaceutical composition for treating cancer comprising the dual target protein.
  • Still another object of the present invention is to provide a cancer diagnostic composition comprising the dual target protein.
  • Another object of the present invention is the conformation of DLL4, comprising amino acid sequence 58 to 65 and amino acid sequence 110 to 115 in the amino acid sequence of the DLL4 (Delta-like ligand 4) protein represented by SEQ ID NO: 21 To provide an epitope.
  • Another object of the present invention is to provide a monoclonal antibody that specifically binds to DLL4, which recognizes the conformational epitope.
  • Still another object of the present invention is to provide a polynucleotide encoding the monoclonal antibody, an expression vector comprising the polynucleotide, and a transformant comprising the expression vector.
  • the dual target protein of the present invention can treat cancer by simultaneously binding to VEGF and DLL4, and by using a novel protein that specifically binds to DLL4, it has excellent binding and anticancer effects, and thus can be widely used in cancer treatment and diagnostic fields.
  • VEGF and DLL4 vascular endothelial growth factor receptor 4
  • a novel protein that specifically binds to DLL4 it has excellent binding and anticancer effects, and thus can be widely used in cancer treatment and diagnostic fields.
  • 1A and 1B show the structure of a dual target protein capable of simultaneously binding DLL4 and VEGF.
  • Figure 2a shows the results confirmed by SDS-PAGE after purification by expressing a dual target protein capable of binding DLL4 and VEGF in CHO cells.
  • FIG. 2b shows the result of SEC-HPLC chromatography analysis after purification by expressing a dual target protein capable of simultaneously binding DLL4 and VEGF in CHO cells.
  • Figure 3 shows the results of analyzing the binding assay for the DLL4 and VEGF of the dual target protein for DLL4 and VEGF (enzyme-linked immunosorbent assay) (ELISA).
  • FIG 4a shows the measurement results of the equilibrium dissociation constant (KD) value for DLL4, the antigen of the dual target protein through the Biacore analysis method.
  • Figure 4b shows the measurement results of the equilibrium dissociation constant (KD) value for the VEGF antigen of the dual target protein via the Biacore analysis method.
  • Figure 5 shows the results of the neutralization assay (Neutralizing assay) for the dual target protein DLL4 and VEGF by ELISA.
  • Figure 6 shows the results of complex formation of human DLL4 and MLCK2 antibody with or without crosslinker.
  • FIG. 7 shows a model in which fragments consisting of amino acid residues 58-65 [FRVCLKHF] and SEQ ID NO: 22 of DLL4 represented by SEQ ID NO: 21 on the surface of a human DLL4 C2 domain (27-174) constitute a continuous molecular surface will be.
  • Figure 8 shows the results confirmed by Western blotting the binding capacity of the mutant protein encoding the deletion fragments of the wild type and the extracellular domain of DLL4.
  • Figure 9a shows the result of confirming that when treated with Avastin, an antibody targeting VEGF, proliferation of vascular endothelial cells is inhibited in a concentration-dependent manner regardless of the presence or absence of DLL4.
  • Figure 9b shows the results confirming that the proliferation of vascular endothelial cells depending on the DLL4 antibody treatment concentration only in the experimental group with DLL4 when the antibody to DLL4 alone.
  • Figure 9c shows that when treated with a double-target protein, the experimental group without the DLL4 showed a proliferative inhibitory effect similar to the Avastin antibody treatment (black bar), the experimental group with the DLL4 present showed a result of reduced vascular growth inhibitory effect compared to the Avastin (White bar).
  • FIG. 10 is a Western blot analysis showing that the dual target protein that binds DLL4 and VEGF shows activity of inhibiting DLL4 / Notch signaling pathway and VEGF / VEGFR signaling pathway of vascular endothelial cells (HUVEC). It is shown.
  • Figure 11 shows that the dual target protein that binds DLL4 and VEGF has a strong anticancer effect in comparison with Avastin in the Avastin-resistant human SCH gastric cancer xenograft model constructed in nude mice. Will be confirmed.
  • Figure 12 shows that the dual target protein binding to DLL4 and VEGF has a strong anticancer efficacy in comparison with Avastin in the Avastin-resistant human A549 lung cancer xenograft model constructed in nude mice. Will be confirmed.
  • the present invention comprises amino acids sequence 58 to 65 and amino acid sequence 110 to 115 in the amino acid sequence of the DLL4 (Delta-like ligand 4) protein represented by SEQ ID NO: 21
  • DLL4 Delta-like ligand 4
  • SEQ ID NO: 21 To provide a dual-target protein comprising a protein that specifically binds to DLL4, which recognizes the conformal epitope of DLL4, and an antibody that specifically binds to Vascular endotelial growth factor (VEGF).
  • VEGF Vascular endotelial growth factor
  • double-target protein refers to a protein capable of binding two different kinds of antigens (target proteins). Specifically, it does not exist naturally, and is preferably in a form prepared by genetic engineering or any method.
  • the dual target protein may bind to VEGF overexpressed in cancer cells and DLL4 expressed in endothelial cells.
  • the dual target protein may be in the form of an antibody.
  • the "dual target protein" of the present invention may be mixed with "dual target antibody", “dual antibody” or “dual antibody protein”.
  • the dual target protein of the present invention may have VEGF and DLL4 as antigens.
  • the form of the double-target protein of the present invention is not particularly limited thereto, and includes a form of a double-target protein in which an antibody specifically binding to VEGF in IgG form and a protein specifically binding to DLL4 are linked by a linker. The structure is as shown schematically in FIG. 1A.
  • the dual target protein of the present invention may specifically include a heavy chain amino acid sequence represented by SEQ ID NO: 1 and a light chain amino acid sequence represented by SEQ ID NO: 20, but is not limited thereto.
  • antibody refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, including polyclonal antibodies, monoclonal antibodies, It includes both full antibodies and antibody fragments.
  • the term also includes forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies).
  • the full length antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by a heavy chain and a disulfide bond.
  • the full length antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype and may include IgG1, IgG2, IgG3 and IgG4.
  • the antibodies may include bivalent, diabodies, triabodies, and tetrabodies.
  • the antibody specifically binding to VEGF of the present invention may be in the form of IgG.
  • the dual target protein is an antibody that specifically binds to Vascular endothelial growth factor (VEGF) in the form of immunoglobulin (Immunoglobulin G, IgG), and a full-length antibody that specifically binds to DLL4 (Delta-like ligand 4), Fab Proteins in the form of ', F (ab') 2 , Fab, Fv, rIgG or scFv may be in linker form.
  • VEGF Vascular endothelial growth factor
  • IgG immunoglobulin G
  • DLL4 Delta-like ligand 4
  • Fab Proteins in the form of ', F (ab') 2 , Fab, Fv, rIgG or scFv may be in linker form.
  • immunoglobulins and scFvs have heavy and light chains, each heavy and light chain comprising a constant region and a variable region (the region is also known as the domain).
  • the variable regions of the light and heavy chains comprise three multivariable regions and four framework regions called complementarity-determining regions (hereinafter referred to as "CDRs").
  • the CDRs mainly serve to bind epitopes of antigens.
  • the CDRs of each chain are typically called CDR1, CDR2, CDR3, starting sequentially from the N-terminus, and can also be identified by the chain where the particular CDR is located.
  • the dual target protein comprising a protein specifically binding to DLL4 and an antibody specifically binding to VEGF shows a strong affinity for human-derived DLL4 and VEGF, and cells expressing DLL4 (eg, cancer cells). Or vascular endothelial cells) effectively inhibits the binding of Notch 1 or Notch 4 receptors, as well as inhibiting neovascularization by vascular endothelial cells expressing VEGF receptors by VEGF overexpressing cancer cells. In the treatment of diseases such as can be expected a stronger therapeutic effect.
  • the antibody specifically binding to VEGF and the protein specifically binding to DLL4 maintain their specific binding, and in particular, because they can simultaneously inhibit two targets (antigens), It may be more effective than inhibiting in combination with the target and may simultaneously inhibit two signals.
  • antibody fragment includes antigen-binding forms of an antibody, including fragments having antigen-binding ability, such as Fab ', F (ab') 2 , Fab, Fv, rIgG and scFv. .
  • the term encompasses single-chain variavle fragments (scFv) and includes bivalent or diabodies, triabodies and tetrabodies.
  • single-chain variavle fragment refers to a minimum antibody fragment having a complete antigen-recognition and antigen-binding site, comprising the VH and VL domains of an antibody, wherein the domains are single polypeptides. May be present in the chain.
  • DLL4 formational epitope of DLL4 comprising amino acids 58-65 and amino acids 110-115 in the amino acid sequence of the DLL4 (Delta-like ligand 4) protein represented by SEQ ID NO: 21
  • Dual-target proteins including proteins that bind specifically to DLL4, which recognizes conformational epitopes, and antibodies that specifically bind to Vascular Endotelial Growth Factor (VEGF) are two signaling pathways by DLL4 and VEGF. Any dual target protein capable of simultaneously inhibiting may be included without limitation.
  • Antibodies that specifically bind to VEGF constituting the dual target protein and antibodies that specifically bind to DLL4 may include both the full-length antibodies and antibody fragments described above.
  • the term “conformational epitope of DLL4 comprising amino acids 58 to 65 and amino acids 110 to 115 in the amino acid sequence of the DLL4 (Delta-like ligand 4) protein represented by SEQ ID NO: 21 protein that specifically binds to DLL4, which recognizes a conformational epitope "is a conformation of DLL4 comprising amino acid sequences 58 to 65 and amino acid sequences 110 to 115 in the amino acid sequence in DLL4 represented by SEQ ID NO: 21 It means a protein that specifically binds to the enemy epitope.
  • Such a protein means a protein capable of inhibiting the growth of cancer and exhibiting a cancer therapeutic effect, and may bind to the epitope with high affinity and neutralize DLL4 activity.
  • the protein may block DLL4 binding to Notch receptors and may inhibit signaling by DLL4.
  • the protein that specifically binds to a conformational epitope comprising SEQ ID NO: 21 and SEQ ID NO: 22 of the DLL4 is specifically a full length antibody, Fab ', F (ab') 2 , Fab, Fv, rIgG or It may be in the form of a single-chain variable fragment (scFv).
  • the protein specifically binding to the DLL4 is a heavy chain CDR1 represented by SEQ ID NO: 2; A heavy chain CDR2 represented by SEQ ID NO: 3; And a heavy chain variable region comprising a heavy chain CDR3 represented by SEQ ID NO: 4 and a light chain CDR1 represented by SEQ ID NO: 5; Light chain CDR2 represented by SEQ ID NO: 6; And a light chain variable region comprising the light chain CDR3 represented by SEQ ID NO: 7.
  • the heavy chain may include a heavy chain amino acid sequence represented by SEQ ID NO: 8
  • the light chain may include a light chain amino acid sequence represented by SEQ ID NO: 9, but includes the CDR sequences described above, If the protein is specifically bound to exhibit a cancer therapeutic effect, the sequence may be different.
  • the heavy and light chains may be linked via a linker.
  • a protein that specifically binds to DLL4 which is a component of the dual target protein of the present invention, may specifically inhibit the interaction between DLL4 and Notch protein while specifically binding to mouse DLL4 as well as human DLL4.
  • the epitope of the antibody specifically binding to DLL4 of the dual target protein of the present invention excellent in the biological inhibitory activity of DLL4 and VEGF.
  • amino acid sequence 58-65 (SEQ ID NO: 22) and / or amino acid sequence 110-115 (SEQ ID NO: 23) of DLL4 may be an epitope of an antibody that specifically binds to DLL4 according to the present invention
  • the molecular surface portion formed by the SEQ ID NOs: 22 and 23 of the DLL4 may be a conformational epitope.
  • delta-like ligand 4 refers to a protein that binds to Notch 1 or Notch 4 receptors, as one of the delta classes of ligands using Notch protein as a receptor. However, it is not limited thereto.
  • the DLL4 may be included without limitation as long as it is a mammalian DLL4. Specifically, the DLL4 may mean a DLL4 of a human or a mouse. DLL4 is overexpressed in various cancer cells, including tumor vasculature, and is known to promote cancer growth by increasing abnormal vascular numbers in several xenograft models.
  • DLL4 (Delta-like ligand 4) protein represented by SEQ ID NO: 21 of the present invention specific for the conformational epitope of DLL4 comprising the amino acid sequence of 58 to 65 and amino acid sequence of 110 to 115 Dual-target proteins, including proteins that bind to can be effectively used in cancer therapy by inhibiting the function of DLL4.
  • the information on the DLL4 may be obtained from a known database such as GenBank of the National Institutes of Health.
  • the accession number may be information of DLL4 having Gene ID: 54567 and NCBI Reference Sequence: NM_019074.3, and the DLL4 may be a sequence number. May comprise the amino acid sequence of 21.
  • notch receptor refers to a protein that mediates Notch signal transduction and may be used in combination with Notch.
  • the notch receptor may be included as long as it is a protein that mediates Notch signal transduction, and specifically, may be Notch 1 or Notch 4 receptor, but is not limited thereto.
  • the term "inhibit the interaction between human delta-like ligand 4 (DLL4) and Notch receptors” means that a dual-target protein that specifically binds to DLL4 of the present invention binds DLL4 between DLL4 and Notch receptors.
  • the dual target protein specific for the conformational epitope of may bind to DLL4 and inhibit the interaction of DLL4 with Notch 1 or Notch 4 receptor, but is not limited thereto.
  • DLL4 (Delta-like ligand 4) protein represented by SEQ ID NO: 21 of the present invention
  • a duplex specific to the conformational epitope of DLL4 comprising amino acids 58 to 65 and amino acids 110 to 115 Interaction between DLL4 and Notch receptors is inhibited by the binding of the target protein, and the structural change of Notch protein due to Notch receptor binding of DLL4 is not brought about so that it cannot be hydrolyzed and thus notch signaling.
  • DLL4 and Notch receptors The binding of DLL4 and Notch receptors in cancer is known to increase blood vessel size, activate signaling between gonocyte endothelial cells or notch signaling between cancer cells and vascular endothelial cells, thereby inhibiting cancer proliferation and metastasis.
  • blocking Notch signaling by DLL4 in cancer is particularly difficult to control angiogenesis, which can inhibit cancer growth.
  • blocking DLL4 results in the deletion of lateral inhibition in the cells at the end of the angiogenesis site, resulting in excessive germination, resulting in an angiogenic reaction that results in excessively high but poor productivity.
  • hypoxia can be induced around the cancer, resulting in anti-cancer effects, especially in cancers that are resistant to anti-VEGF treatment.
  • dual target proteins comprising proteins that specifically bind to DLL4 of the present invention that effectively inhibit the interaction between DLL4 and Notch can be effectively used in the treatment of cancer.
  • antibody that specifically binds VEGF includes any antibody that specifically binds to VEGF as an antigen in cancer cells.
  • Specific examples may include, but are not limited to, Bevacizumab (trade name Avastin ® ), a therapeutic antibody targeting VEGF.
  • Antibodies that specifically bind to such VEGF include all the forms of the full-length antibodies or antibody fragments described above, and may be in the form of IgG antibodies, but are not limited thereto.
  • VEGF is a ligand that plays an important role in angiogenesis. When it is suppressed, angiogenesis is not achieved and cancer can be treated.
  • Bevacizumab is a therapeutic antibody that can be stably used as approved by the US FDA as Avastin of Genentech.
  • the heavy chain variable region is a heavy chain CDR1 represented by SEQ ID NO: 10; A heavy chain CDR2 represented by SEQ ID NO: 11; And a heavy chain CDR3 represented by SEQ ID NO: 12 and the light chain variable region comprises a light chain CDR1 represented by SEQ ID NO: 13; Light chain CDR2 represented by SEQ ID NO: 14; And light chain CDR3 represented by SEQ ID NO: 15, and more specifically, may include a heavy chain amino acid sequence represented by SEQ ID NO: 16 and a light chain amino acid sequence represented by SEQ ID NO: 17, but specifically binds to VEGF.
  • the sequence of the protein that can exhibit a cancer therapeutic effect can be included without limitation.
  • Antibodies that specifically bind to VEGF which is a component of the dual target protein of the present invention, specifically bind to VEGF overexpressed in cancer cells, so that the dual target protein of the present invention can be concentrated on cancer cells expressing VEGF. In combination with VEGF, it may have anticancer activity by itself.
  • vascular endothelial growth factor is a growth factor that enhances the growth activity of vascular endothelial cells, secreted by various cells such as dashed cells, smooth muscle cells, tumor cells, and the like. . Not only plays an important role in the generation of angiogenesis, but also to induce angiogenesis (angiogenesis) for the supply of oxygen in the tumor tissue that is fast growth and metabolism.
  • angiogenesis angiogenesis
  • the pathway by the VEGF protein and its receptor has been studied in adult as a target signaling pathway of anticancer drugs.
  • the VEGF binding site of the double-target protein means inhibiting the interaction between human VEGF and the VEGF receptor, specifically, the VEGF-specific double-target protein binds to VEGF and interacts with the VEGF and VEGFR-2 receptors. It may mean to inhibit the action, but is not limited thereto.
  • the VEGF receptor may be included as long as it is a protein that binds to VEGF in mammals, but may specifically mean a protein that binds to human VEGF.
  • VEGF / VEGF receptor signaling by VEGF receptor binding of VEGF through inhibition of the interaction between VEGF and the VEGF receptor by the dual target protein specific for VEGF of the present invention.
  • VEGF / VEGF receptor signaling is activated in stromal / endothelial cells of cancer tissues, which is different from the mechanism of action of DLL4 / Notch signaling pathways. It strongly inhibits the number of blood vessels, and is known to inhibit the growth and metastasis of cancer by weakening the vascular function (tumor function) in the tumor.
  • the dual target protein specific for DLL4 and VEGF of the present invention can be used as a therapeutic agent having a superior anticancer ability by showing the ability to inhibit neovascularization of cancer tissues of different mechanisms.
  • the dual target protein may be in a form in which a protein specifically binding to DLL4 and an antibody specifically binding to VEGF in IgG (Immuniglobulin G) form are linked by a linker.
  • IgG immunoglobulin G
  • linker basically refers to two different fusion partners (e.g., biological polymers, etc.) that are hydrogen bonds, electrostatic interactions, van der Waals forces, disulfide bonds, salt bridges, It refers to a linker that can be linked using hydrophobic interactions, covalent bonds, and the like, specifically, at least one disulfide bond under physiological conditions or other standard peptide conditions (eg, peptide purification conditions, peptide storage conditions).
  • a hinge that can have at least one cysteine that can participate and, in addition to simply connecting each fusion partner, serves to provide a certain amount of spacing between the fusion partners or to provide flexibility or rigidity to the fusion. Can play the role of).
  • the linker may be a non-peptide linker or a peptide linker, and may include all directly linked by peptide bonds, disulfide bonds, and the like.
  • the linker is not particularly limited thereto, but may be a polypeptide capable of linking a protein specifically binding to DLL4 and an antibody specifically binding to VEGF, and more specifically, to the DLL4. It may be a peptide linker capable of linking the C-terminus of the Fc region of the antibody that specifically binds to VEGF and the protein that binds specifically, and more specifically, the GGGGS motif is an amino acid sequence of three repeats. It may be a constructed peptide linker. The GGGGS motif may be repeated 1 to 10 times, and most specifically, may be composed of an amino acid sequence encoded by the following amino acid sequence of SEQ ID NO: 18 or polynucleotide sequence of SEQ ID NO: 19.
  • Linker Peptide (SEQ ID NO: 18): GGGGSGGGGSGGGGS
  • non-peptide linker refers to a biocompatible linker having two or more repeating units linked thereto, and the repeating units may be linked to each other through any covalent bonds other than peptide bonds.
  • Non-peptide linkers of the present invention are polyethylene glycol (PEG) homopolymers, polypropylene glycol homopolymers, ethylene glycol-propylene glycol copolymers, polyoxy ethylenated polyols, polyvinyl alcohols, polysaccharides, dextran, poly Biodegradable polymers such as vinyl ethyl ether, lipid polymers, chitin, hyaluronic acid or combinations thereof.
  • PEG polyethylene glycol
  • polypropylene glycol homopolymers ethylene glycol-propylene glycol copolymers
  • polyoxy ethylenated polyols polyvinyl alcohols
  • polysaccharides polysaccharides
  • dextran polysaccharides
  • poly Biodegradable polymers such as vinyl ethyl ether, lipid polymers, chitin, hyaluronic acid or combinations thereof.
  • it may be a polyethylene glycol homopolymer,
  • it may be a polyethylene glycol homopolymer having a molecular weight of 1 to 5 kDa, and most specifically, a protein and VEGF that specifically bind to DLL4 in the form of bifunctional aldehyde at both ends of about 3.4 kDa. It may be a linker capable of linking an antibody that specifically binds. In particular, having a reactor of reactive aldehyde groups at both ends is effective for minimizing nonspecific reactions.
  • Sites directly or indirectly linked through the linker are not particularly limited thereto, and may be Fc moieties, Fab ′, F (ab ′) 2 , Fab, Fv, and the like.
  • the dual target protein is not particularly limited thereto, but all or a portion of a protein specifically binding to DLL4 and a form in which all or a portion of an antibody specifically binding to VEGF is linked; Alternatively, all or a portion of a protein that specifically binds to DLL4 and all or a portion of an antibody specifically binding to VEGF may be linked by a peptide linker.
  • all or a portion of the protein that specifically binds to DLL4 and all or a portion of the heavy chain of the antibody that specifically binds to VEGF is linked by a peptide linker; All or a portion of a protein that specifically binds to DLL4 and a form in which all or a portion of the light chain of the antibody specifically binds to VEGF is linked with a peptide linker; Or a combination thereof.
  • the inventors insert a polynucleotide encoding a double target protein in which a heavy chain region C-terminus of Avastin in IgG form and a DLL4 binding protein in scFv form by linker are linked to a vector, and the animal cell
  • the Avastin-DLL4 binding double target protein was isolated to introduce a double target protein Avastin-DLL4 BsAb that specifically binds DLL4 and VEGF.
  • the dual target protein molecule has a structure in which an Avastin IgG antibody molecule and a DLL4 binding scFv are linked by a linker (FIG. 1).
  • Avastin-DLL4 binding dual target protein introduced and expressed in the animal cells was isolated and confirmed for expression and purity (FIGS. 2A and 2B).
  • the Avastin-DLL4 binding dual target protein specifically binds the target VEGF and DLL4 (FIG. 3).
  • the double-target protein showed similar binding activity to the control antibody for each antigen, showing a KD value of 30 nM for human DLL4 and a KD value of 0.126 nM for human VEGF (Tables 2-3). It was confirmed that each signal transduction pathway by the binding of endothelial cells to DLL4 and human Notch 1 receptors and the binding of VEGF and VEGF receptors was effectively inhibited by dual target protein treatment (FIG.
  • the present invention provides a polynucleotide encoding the dual target protein, an expression vector comprising the polynucleotide, and a transformant into which the expression vector is introduced.
  • Expression vectors comprising the polynucleotide encoding the dual target protein provided in the present invention is not particularly limited, mammalian cells (eg, human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells May be a vector capable of replicating and / or expressing the polynucleotide in eukaryotic or prokaryotic cells, including yeast cells, insect cells or bacterial cells (eg, Escherichia coli, etc.), specifically in the host cell It may be a vector operably linked to an appropriate promoter for expression of the polynucleotide, and may comprise a vector comprising at least one selection marker, more specifically phage, plasmid, cosmid, mini-chromosome, virus, retroviral vector It may be a form in which the polynucleotide is introduced.
  • mammalian cells eg, human, monkey, rabbit, rat, hamster, mouse cells, etc.
  • plant cells May be a vector capable of replicating
  • the expression vector comprising the polynucleotide encoding the double target protein is an expression vector comprising both a polynucleotide encoding the heavy or light chain of the double target protein or an expression vector including all of the polynucleotides encoding a heavy or light chain. Can be.
  • the transformant introduced with the expression vector provided by the present invention is not particularly limited thereto, but the bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium transformed by introducing the expression vector; Yeast cells; Fungal cells such as Pchia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; Chinese hamster ovary cells (CHO), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK ( Animal cells such as baby hamster kidney cells, baby hamster kidney cells, HEK (human embryonic kidney cells), and PERC.6 (human retinal cells); Or plant cells.
  • CHO-S cells were used as host cells.
  • introduction refers to a method of delivering a vector comprising a polynucleotide encoding a dual target protein to a host cell.
  • introductions include calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroshock, microinjection, liposome fusion, lipofectamine and protoplast fusion. It can be carried out by various methods known in the art.
  • transduction refers to the delivery of a target product into cells using viral particles by means of infection.
  • the vector can be introduced into the host cell by gene bombardment or the like. Introduction in the present invention can be used interchangeably with transformation.
  • the present invention provides a method for preparing the dual target protein.
  • step (a) culturing the transformant to produce a dual target protein; And (b) recovering the dual target protein produced in step (a), which comprises a protein that specifically binds to DLL4 and an antibody that specifically binds to Vascular Endotelial Growth Factor (VEGF). It may be a method for producing a target protein.
  • VEGF Vascular Endotelial Growth Factor
  • the polynucleotide and heavy chain variable region encoding an antibody that specifically binds VEGF include heavy chain CDR1 represented by SEQ ID NO: 2; A heavy chain CDR2 represented by SEQ ID NO: 3; And a heavy chain CDR3 represented by SEQ ID NO: 4 and the light chain variable region comprises a light chain CDR1 represented by SEQ ID NO: 5; Light chain CDR2 represented by SEQ ID NO: 6; And obtaining a polynucleotide encoding a protein that specifically binds to DLL4, including the light chain CDR3 represented by SEQ ID NO: 7; (b) a poly encoding the protein specifically binding to the 3'-terminus of the polynucleotide encoding the Fc region and the DLL4 of the polynucleotide encoding the antibody specifically binding to the VEGF obtained in step (a) Linking the 5'-terminus of a nucleotide with a linker to obtain a polynucle
  • the preparation method (a) the polynucleotide encoding the antibody specifically binding to VEGF and the heavy chain variable region is a heavy chain CDR1 represented by SEQ ID NO: 2; A heavy chain CDR2 represented by SEQ ID NO: 3; And a heavy chain CDR3 represented by SEQ ID NO: 4 and the light chain variable region comprises a light chain CDR1 represented by SEQ ID NO: 5; Light chain CDR2 represented by SEQ ID NO: 6; And obtaining a polynucleotide encoding a protein that specifically binds to DLL4, including the light chain CDR3 represented by SEQ ID NO: 7; (b) cloning the polynucleotide of step (a) to prepare an expression vector; (c) culturing the transformant by introducing the expression vector of step (b) into a host cell; And (d) obtaining an antibody and a protein binding to DLL4 specifically binding to VEGF from the transformant of step (c), to the C-terminus and D
  • the dual target protein of the present invention can be prepared by any of the above known recombinant means or biochemical methods, and the antibody can be introduced into an appropriate host cell and recovered from the culture of the transformant.
  • dual-target proteins can be separated by known separation methods, such as conventional immunoglobulin purification procedures such as protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. It can be properly separated from the culture medium by, but is not limited thereto.
  • the present invention provides a composition comprising the dual target protein.
  • the present invention provides a composition for treating cancer comprising the dual target protein.
  • the dual target protein may be involved in inhibiting cancer growth by simultaneously binding DLL4 and VEGF to inhibit Notch and binding to VEGF receptors.
  • the DLL4 / Notch receptor and the VEGF / VEGF receptor are as described above.
  • the composition of the present invention can be administered in vivo a composition comprising a dual target protein that specifically binds DLL4 and VEGF, to inhibit the development, proliferation or metastasis of the cancer or to prevent the cancer to be treated.
  • cancer in the present invention includes, without limitation, the type of cancer, for example esophageal cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovary Cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma or multiple myeloma hematologic cancer.
  • treatment may mean any action that improves or advantageously changes the symptoms of cancer by administration of the composition.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” refers to a carrier or diluent that does not irritate an organism and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers in compositions formulated as liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary.
  • diluents may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • the pharmaceutical composition may be in various oral or parenteral formulations.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
  • Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin.
  • lubricants such as magnesium stearate, talc and the like are also used.
  • Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • the pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It can have one formulation.
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is determined by the type and severity, age, sex, disease of the individual. It may be determined according to the type, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field.
  • the compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.
  • the dual target protein of the present invention binds to both VEGF and DLL4 (FIGS. 3, 4A and 4B), can neutralize DLL4 (FIG. 5), and human gastric cancer with Avastin resistance.
  • SCH human gastric cancer with Avastin resistance
  • lung cancer A549) cell line xenograft model confirmed the anti-cancer efficacy of the dual target protein (Figs. 11 and 12), it was confirmed that it can be used as an active ingredient for cancer treatment compositions.
  • the present invention provides a method for treating cancer using a pharmaceutical composition comprising the dual target protein.
  • the method may be achieved by administering the pharmaceutical composition in a pharmaceutically effective amount.
  • the dual target protein and the pharmaceutically effective amount are as described above.
  • the dual target protein may be a method for treating cancer, comprising administering a pharmaceutical composition further comprising a pharmaceutically acceptable carrier to an individual with or suspected of having cancer, and which may be used as a carrier and
  • the cancer is the same as described above.
  • the subject may include a mammal, a bird, etc., including a cow, a pig, a sheep, a chicken, a dog, a human, and the like, and the subject to which the cancer is treated by administration of the composition of the present invention may be included without limitation.
  • the composition may be administered in the form of a liquid, powder, aerosol, capsule, enteric skin tablets or capsules or suppositories.
  • Routes of administration include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, endothelial, oral, topical, nasal, pulmonary, rectal, and the like.
  • the oral composition needs to be formulated to coat the active agent or protect it from degradation in the stomach.
  • the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.
  • the present invention provides a cancer diagnostic composition comprising the dual target protein.
  • the dual target protein and cancer are as described above.
  • diagnosis means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether the cancer has developed.
  • the cancer diagnostic composition of the present invention measures the level of VEGF or DLL4 protein in an isolated sample of a subject suspected of cancer by using the dual target protein of the present invention, and thus the level of the measured VEGF or DLL4 protein is higher than that of a normal control sample. Can be used to determine cancer.
  • Methods for measuring protein levels include Western blot, Enzyme Linked Immunosorbent Assay (ELISA), badioimmunoassay (otA: badioimmunoassay), radioioimmunodiffusion, and Ouchterlony immunity. Diffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS and protein chip, but are not limited thereto. Through such analytical methods, VEGF or DLL4 protein levels can be compared between a normal control group and a suspected cancer patient, thereby enabling diagnosis of cancer in a suspected cancer patient.
  • composition for diagnosing cancer of the present invention may further include, without limitation, what is known in the art as required to perform a method for measuring the level of the protein in addition to the dual target protein of the present invention.
  • the present invention provides a method for treating cancer, comprising: (a) measuring the level of VEGF or DLL4 protein in an isolated sample of an individual suspected of cancer using the dual target protein; And (b) provides a method for diagnosing cancer, comprising the step of judging cancer when the level of VEGF or DLL4 protein measured in step (a) is higher than the normal control.
  • the steps (method) of measuring the dual target protein, cancer, subject, diagnosis and protein are as described above.
  • sample includes samples, such as whole blood, serum, blood, plasma, saliva, urine, sputum, lymph, cerebrospinal fluid, and intercellular fluid, which differ in expression levels of VEGF or DLL4 in cancer patients. It is not limited.
  • the present invention provides a three-dimensional structure of DLL4, comprising amino acid sequence 58 to 65 and amino acid sequence 110 to 115 in the amino acid sequence of the DLL4 protein (Delta-like ligand 4) represented by SEQ ID NO: 21 Provide morphological epitopes.
  • the crosslinking reaction and mass spectrometry have identified the amino acid residues in which the crosslinking reaction occurs in DLL4 of SEQ ID NO: 21, amino acid sequence 58 to 65 containing the residues [FRVCLKHF] and 110 Two fragments of amino acid sequence from SEQ ID NO: 115 (SEQ ID NO: 23) were confirmed to form epitopes of DLL4 by constructing a continuous molecular surface, as shown in FIG.
  • the present invention provides a monoclonal antibody that specifically binds to DLL4, which recognizes the conformational epitope.
  • the monoclonal antibody is a heavy chain CDR1 represented by SEQ ID NO: 2; A heavy chain CDR2 represented by SEQ ID NO: 3; And a heavy chain variable region comprising a heavy chain CDR3 represented by SEQ ID NO: 4 and a light chain CDR1 represented by SEQ ID NO: 5; Light chain CDR2 represented by SEQ ID NO: 6; And a light chain variable region comprising the light chain CDR3 represented by SEQ ID NO: 7. More specifically, the heavy chain may be composed of the amino acid sequence of SEQ ID NO: 8, the light chain may be composed of the amino acid sequence of SEQ ID NO: 9.
  • the present invention provides a polynucleotide encoding the monoclonal antibody, an expression vector comprising the polynucleotide and a transformant into which the expression vector is introduced.
  • DLL4 monoclonal antibody, vector, transformant and the like of the present invention are the same as described above.
  • the present invention provides a method for treating cancer, comprising administering the dual target protein to a subject suspected of having cancer.
  • the subject may be a subject in need of preventing or treating cancer, and may be a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a antelope, a dog, a cat, etc., in need of treatment of cancer and similar symptoms. However, it is not limited thereto.
  • the term "administration" refers to introducing the pharmaceutical composition of the present invention to a patient in any suitable manner, the route of administration of the composition of the present invention being oral or parenteral as long as it can reach the target tissue. Administration can be via a variety of routes.
  • the method of treating cancer of the present invention comprises administering a dual target protein or a pharmaceutical composition comprising the same in a therapeutically effective amount. It will be apparent to those skilled in the art that a suitable total daily usage may be determined by the practitioner within the scope of good medical judgment. It may also be administered once or in divided doses.
  • a specific therapeutically effective amount for a particular patient is determined by the specific composition, including the type and severity of the reactions to be achieved, whether or not the other agent is used in some cases, the age, weight, general state of health of the patient, It can be applied differently depending on various factors and similar factors well known in the medical field, including sex and diet, time of administration, route of administration and rate of composition, duration of treatment, drugs used with or concurrent with the specific composition.
  • Example 1 Preparation of anti DLL4 / VEGF dual target protein
  • the extracellular domain antigen of human DLL4 was a human DLL4 protein (Cat: 1506-D4 / CF) provided by R & D Systems. This DLL4 antigen protein was identified as Accession No. Amino acids 27-524 of the DLL4 amino acid sequence of Q9NR61. The histidine-tag (10-His tag) is ligated at the protein C-terminus.
  • Antigens against specific regions of another DLL4 extracellular domain were prepared. This particular region comprises amino acids 27 to 251. This region contains a motif called the "DSL (Delta / Serrate / lag-2)" domain that is known to bind the Notch1 receptor.
  • DLL Delta / Serrate / lag-2
  • Mammalian expression plasmid vectors containing a CMV promoter upstream of a polynucleotide encoding a deletion fragment (amino acids 27-251) of the extracellular domain of DLL4 fused to an Fc protein were prepared using standard recombinant DNA techniques. Additional constructs encoding deletion fragments of the chimeric DLL4 of human DLL4 fused to the Fc protein were prepared using conventional recombinant DNA techniques.
  • the constructed construct was transiently transfected into HEK 293E cells to express recombinant fusion proteins comprising human DLL4 amino acids 27 to 251 fused to Fc protein.
  • Conditioning medium was collected every 72 hours to obtain the protein and this was repeated four times. Purification from this conditioning medium was carried out using Protein A affinity chromatography.
  • helper phage was infected to infect 2X YT CMK (2X YT CM, kanamycin (sigma, K1876) 70 ⁇ g / ml, 1 mM IPTG (ELPISBIO, IPTG025)).
  • the medium was incubated at 30 ° C. for 16 hours.
  • the cultured cells were centrifuged (4500 rpm, 15 minutes, 4 ° C), and then dissolved in the supernatant by adding 4% PEG (Fluka, 81253) 6000 and 3% NaCl (sigma, S7653) to ice for 1 hour. Reacted.
  • Nonspecifically bound phages were washed five times with PBS-T (Phosphate buffered saline-0.05% Tween 20) solution, and the remaining antigen-specific phage antibodies were recovered using 100 mM triethylamine solution.
  • PBS-T Phosphate buffered saline-0.05% Tween 20
  • the recovered phages were neutralized with 1M Tris buffer (pH 7.4) and then infected with ER2537 Escherichia coli for 1 hour at 37 ° C., and infected E. coli were plated in LB (Luria-Bertani) agar medium containing carbenicillin overnight at 37 ° Incubated. The next day, E. coli cultured was suspended in 4 mL of SB (superbroth) -carbenicillin medium, 15% glycerol was added, and some were stored at -80 ° C. % Glucose solution (glucose) was added and incubated at 37 °C.
  • the culture medium When the absorbance of the culture medium reached 0.6 at 600 nm (OD 600 ), the culture medium was removed by centrifugation, suspended again in 20 mL of SB-carbenicillin culture medium, and then stirred in a 10 12 PFU VCSM13 helper phage and slowly stirred at 37 ° C. Incubated. The next day, the culture medium was centrifuged, and only the culture medium was taken out, 4% polyethylene glycol 8000 (PEG8000) and 3% sodium chloride (NaCl) were added thereto, precipitated at 4 ° C. for 30 minutes, and then centrifuged. The supernatant was removed and the precipitated phage was suspended in 1 mL of PBS and used as a library to amplify / concentrate the antigen specific clones by repeating the panning process above.
  • PEG8000 polyethylene glycol 8000
  • NaCl sodium chloride
  • coli was suspended in TES solution (Tris, EDTA, sucrose), left at 4 ° C for 1 hour and centrifuged to extract periplasm, which was then used to confirm the binding of recombinant human DLL4 antigen to scFv using ELISA. Used.
  • TES solution Tris, EDTA, sucrose
  • Bound scFv was detected using HRP (Horseradish peroxidase) -anti-HA antibody and tetramethylbenzidine (TMB) substrate.
  • HRP Haseradish peroxidase
  • TMB tetramethylbenzidine
  • Antigen-specific antibody clones identified therefrom were analyzed by sequencing. The results of analyzing the sequence of the selected scFv are shown in Table 1 below.
  • the anti-DLL4 antibody having this sequence was named "MLCK-2”.
  • the scFv type antibody that binds to human DLL4 prepared in Examples 1-3 was linked to Avastin IgG form using a linker to prepare a dual target protein expression vector capable of binding to human VEGF (FIG. 1B).
  • the prepared double target protein has a heavy chain amino acid sequence of SEQ ID NO: 1 (VEGF-DLL4 BsAb heavy chain) and a light chain amino acid sequence of SEQ ID NO: 20.
  • the heavy chain is a heavy chain CDR1 represented by SEQ ID NO: 2; A heavy chain CDR2 represented by SEQ ID NO: 3; And a heavy chain variable region comprising a heavy chain CDR3 represented by SEQ ID NO: 4, wherein the light chain comprises a light chain CDR1 represented by SEQ ID NO: 5; Light chain CDR2 represented by SEQ ID NO: 6; And a light chain variable region comprising the light chain CDR3 set forth in SEQ ID NO: 7.
  • a dual-target protein expression vector in CHO cells 200 cells per bottle in a 500 ml culture Erlenmeyer flask (Corning Costa) was used for the production of suspended cell cells transfected with the gene using a polymer that enhances the efficiency of intracellular gene transfer. A total of 1 L was incubated with mL. 1 L of a mixture of RPMI medium (Invitrogen Corporation) and CHO cell medium containing ultra low IgG Fetal Bovine Serum (Invitrogen Corporation) is added to the cell culture medium (Sanyo) for 4 days. Cultured at to produce recombinant protein. Cell cultures were obtained and centrifuged to separate supernatants containing suspended cells and secreted recombinant protein and filtered once with a 0.22 ⁇ m vacuum filter device (Millipore).
  • Avastin-DLL4 BsAb dual target antibody was purified from the culture primarily using recombinant Protein-A Sepharose column (Hitrap MabSelect Sure, 5 mL, GE healthcare). Specifically, the filtered culture medium was loaded onto a recombinant protein-A Sepharose column. The column was washed with 20 mM column volume with 50 mM Tris-Cl (pH 7.5), 100 mM NaCl, and impurities were washed with 10 mM column volume of 50 mM Na-citrate buffer solution (pH 5.0). The antibody was eluted with 5 mM Na-citrate 10 mM NaCl buffer solution (pH 3.4) and neutralized with 1M Tris-HCl buffer solution (pH 8.0).
  • Fractions separated and purified using the column were analyzed by SDS-PAGE (FIG. 2), and the positive fractions were collected and concentrated in a centrifugal concentrator (Amicon Ultra, Amicon Ultra, 30,000 MWCO, Millipore). Buffer exchange and concentration were performed with phosphorylated formulation buffer solution using the same centrifugal concentrator. Finally, the antibody was sterile filtered with a syringe filter of 0.22 ⁇ m pore diameter, and the absorbance (A 280 ) was measured to determine antibody concentration.
  • the binding capacity of the dual target protein was assessed using an ELISA based solution color development test for the anti DLL4, anti VEGF dual target protein.
  • 96-well plates (Nunc-Immuno Plates, NUNC, Rochester, NY) were prepared at 50 ng / ml for HVEGF (R & D systems, cat: 293-VE) at 50 ng overnight and at 200 ng / ml for rhDLL4 (R & D systems, cat: 1506-D4 / CF) was coated with 100 ⁇ l per well, and the nonspecific binding site was blocked with BSA for 2 hours.
  • Antibodies were diluted 1/5 at 128 nM and 64 nM on 96-well microtiter plates and inoculated with 100 ⁇ l of hDLL4 and hVEGF protein-coated plates, followed by 2 hours of incubation and 0.05% tween 20 Washed 5 times with PBS containing 20). To detect the antibody bound to the plate, HRP conjugated anti Fab antibody (Pierce, cat: 31414) was diluted 1: 40000, treated in a washed 96-well plate, and then reacted at 37 ° C. for 1 hour.
  • Color development was carried out using a colorimetric substrate (3,3 ', 5,5'-Tetramethylbenzidine; Sigma-Aldrich) and the enzyme reaction was stopped with 0.5 mol / l sulfuric acid. Absorbance was measured at a wavelength of 450 nm using a SpectraMax 190 (molecular Devices) instrument.
  • Example 1 the separated and purified dual target protein (double antibody) was named Avastin-DLL4 BsAb, and the affinity for the antigen of the isolated and purified antibody was analyzed as follows. A BIACORE assay was performed to determine the difference in binding capacity of Avastin-DLL4 BsAb dual target antibody to DLL4 and VEGF.
  • the first and third flow paths were blank, the second flow path was fixed as hVEGF, and the fourth flow path was designated as hDLL4.
  • the first and third flow passages are reference values showing nonspecific binding and buffer changes.
  • the experimental results used Fc2-Fc1 and Fc4-Fc3.
  • the Avastin-DLL4 BsAb double target antibody was converted to molar concentration, diluted to 100 nM with running buffer, and then diluted 1/2 consecutively at 5 concentration intervals.
  • the analytical sample was prepared in high purity / concentration so as to have a minimum dilution multiple of 100 or more to minimize the buffer effect. All analyzes were performed in multiples of one sample using the Wizard program, with a regeneration step between all assays to ensure that the baseline of the experiment was constant.
  • the results were analyzed by Biaevaluation software 4.0 version.
  • the baseline was set to 0, the buffer injection (analyte 0 nM) was subtracted from the entire sensorgram, and the results were analyzed using the binding affinity model using the Bivalent binding model.
  • the analysis items are K a (M ⁇ 1 s ⁇ 1 ), Kd (s ⁇ 1 ), and KD (M).
  • K a is an association constant indicating affinity
  • K d is a dissociation constant indicating stability.
  • Table 2 shows the results of the binding capacity analysis for hVEGF of the dual target protein
  • Table 3 shows the results of the binding capacity for hDLL4 of the dual target protein.
  • the neutralizing effect of the Avastin-DLL4 BsAb dual target antibody was assessed through ELISA-based solution competition experiments.
  • 500 ⁇ l / mL of hNotch-1-hFc protein (R & D systems) diluted in PBS was added 100 ⁇ l per well and then overnight at 4 ° C. After coating, the cells were treated with BSA for 2 hours to block nonspecific binding sites.
  • antigenic protein human DLL4-His, 600 ng / mL
  • the plate was then incubated for 2 hours and washed 5 times with PBS containing 0.05% tween 20, and the DLL4 antigen bound to the plate was HRP-conjugated His anti-mouse IgG polyclonal antibody reagent ( The HRP-conjugated His anti-mouse IgG polyclonal antibody was treated in a washed microtiter plate in a ratio of 1: 800 for detection by Roche applied science, and then reacted at 37 ° C. for 1 hour. Then, color development was carried out using a colorimetric substrate (3,3 ', 5,5'-Tetramethylbenzidine; Sigma-Aldrich co.) And the enzyme reaction was stopped with 0.5 mol / l sulfuric acid.
  • a colorimetric substrate (3,3 ', 5,5'-Tetramethylbenzidine; Sigma-Aldrich co.
  • the double-target protein of the present invention has a low IC 50 value of 1.12nM for DLL4, it was confirmed to have a DLL4 inhibitory activity as well as the anti-DLL4 antibody alone.
  • the antigen protein human delta-like ligand 4 (human DLL4, hDLL4, R & D Systems) and the MLCK2 antibodies of Examples 1-3 were mixed in a molar ratio of 2: 1, followed by a K200 crosslinker (CovalX AG.) With a final concentration of 0.2 mg / ml. I put it to be. The mixture was reacted for 3 hours at room temperature to form an antigen-antibody complex, followed by analyzing the molecular weight of the reaction product using Ultraflex II MALDI ToF (Bruker Daltonics) equipment. As shown in FIG.
  • the undeuterium-labeled deuccinimidyl suberate (d0-DSS) and the 12 deuterium-labeled d12-DSS were mixed 1: 1 and dissolved in DMF to prepare a 2 mg / ml solution.
  • Reactants were denatured by reduction and alkylation using dithiothreitol (DTT) and iodoacetamide (DTT) for effective degradation reactions, and fragmented using proteolytic enzymes such as trypsin, alpha-chymotrypsin, and ASP-N protease, respectively.
  • DTT dithiothreitol
  • DTT iodoacetamide
  • proteolytic enzymes such as trypsin, alpha-chymotrypsin, and ASP-N protease, respectively.
  • the resulting fragmentation reactants were subjected to mass spectrometry using the Ultimate 3000 nano-liquid chromatography system (Dionex) and the LTQ Orbitrap XL mass spectrometer (Thermo).
  • the resulting mass spectrometry data were analyzed using Xquest (version 2.0) software, Stavrox (version 2.1) software.
  • Crosslinked peptide pairs were fractionated. As a result,
  • the crosslinking reactions on human DLL4 occurred at amino acids residues 59, 63, 64, and 110 and included [58-65, FRVCLKHF] (SEQ ID NO: 22) and [110-115, TWPGTF] (SEQ ID NO: 23).
  • the two fragments constitute a contiguous molecular surface as shown in FIG. 7 on the human DLL4 C2 domain (27-174) model, and these two sequences could be estimated as epitopes of human DLL4 against MLCK2 antibodies.
  • a panel of alanine substitution mutants of human DLL4 was prepared as follows, in which the amino acids 64 (Histidine, histidine), 65 (phenylalanine, phenylalanine) and 69 (Valine, valine) of the human DLL4 extracellular protein region were prepared, respectively.
  • the amino acid of was substituted with alanine (Alanine).
  • a vector used for preparing an antigen for a specific region of the DLL4 extracellular domain was used as shown in Example 1-1.
  • the vector is a vector comprising genes of amino acids 27 to 251 of the human DLL4 specific region, which region is known as "DSL (delta / serrate / lag-2)" domain that binds to the Notch1 receptor. It contains a motif called.
  • a mammalian expression plasmid vector comprising a CMV promoter upstream of a polynucleotide encoding a deletion fragment (amino acids 27-251) of the extracellular domain of DLL4 fused to an Fc protein.
  • Recombinant DNA technology QuikChange Site-Directed Mutagenesis, Agilent
  • Transfection was performed using (Invitrogen, Invitrogen) and cultured for 4 days to obtain an expression medium.
  • a protein encoding a deletion fragment (amino acids 27 to 251) of the extracellular domain of wild type DLL4 was used.
  • the mutant expression medium incubated for 4 days was centrifuged at 1000 rpm for 10 minutes at room temperature to remove suspended solids, followed by 0.45 ⁇ m syringe filtration.
  • Western block was quantified using the Octet ® system (ForteBio) the expression level of the mutant expression medium for ratting experiments, so as to produce a constant amount of protein during SDS-Gel Loading use them. Thereafter, 20 ⁇ l of mutant expression medium was loaded onto two gels in the same manner as Novex 4-12% Bis / Tris gel, and gel electrophoresis was performed at 140V for 50 minutes using MOPS buffer.
  • a protein encoding a deletion fragment (amino acids 27 to 251) of the extracellular domain of wild-type DLL4 was used. After electrophoresis, protein bands were transferred to polyvinylidene difluoride membrane. There are two methods in total, one of which is HRP-conjugated anti-human to confirm that the SDS-Gel loading of deletion fragments (amino acids 27-251) of the extracellular domain of DLL4 was loaded with a certain amount of mutant and wild-type proteins.
  • the Fc antibody (1: 10000) (Pierce Cat: 31413) was used to bind to the transferred membrane, and then washed three times with PBS-T. The other was to determine the binding capacity of the MLCK2 antibody to the mutant.
  • Example 7 Analysis of the effect of DLL4 / VEGF double antibody on vascular endothelial cell (HUVEC) proliferation
  • vascular endothelial cell (HUVEC) proliferation of the double antibody binding to DLL4 and VEGF
  • human umbilical vein endothelial cells (HUVEC) were purchased from Lonza and used in the experiment.
  • the culture of HUVEC was coated with T-flask (Nunc) at room temperature for 4-6 hours with PBS buffer solution (Gibco) in which 1% gelatin (Sigma) was dissolved and washed with PBS.
  • the medium used was EBM-2 (Lonza) containing EGM-2 Single Quot (Lonza), and the culture of cells was subcultured in a 37 ° C., 5% CO 2 incubator at a density not exceeding 80%. Experiments were performed using cells within passage 6.
  • the vascular endothelial cell proliferation assay was performed as follows. To prepare hDLL4 coated plates, rhDLL4 (R & D systems) was diluted to 1 mg / mL using Carbonate buffer solution in 96-well plates (BD) the day before the experiment, and then inoculated at 100 mL / well to stand overnight at 4 ° C. I made it. In addition, HUVEC was to be left in EBM-2 medium with 0.1% FBS for 24 hours to minimize the effect of serum.
  • hVEGF 50 ng / mL
  • antibody Avastin: 20 mg / mL
  • DLL4 alone antibody 20 mg / mL
  • Avastin for each experimental group -DLL4 BsAb dual antibody: 26 mg / mL
  • HUVECs starved for 24 hours from the day before were single-celled, diluted to 4 x 10 3 cells / well using EBM-2 minimal medium and inoculated into wells treated with antibodies in a 37 ° C., 5% CO 2 incubator.
  • cell counting kit-8 (CCK-8, Dojino) was treated with 10 mL in each well and allowed to stand for 5 hours in a 37 °C, 5% CO 2 incubator. Absorbance was measured at a wavelength of 450 nm using a SpectraMax 190 (molecular Devices) device to compare the proliferation of cells in each group (FIG. 9).
  • the effect of inhibiting the proliferation of the VEGF-only antibody treatment can be interpreted as the experimental result that the double antibody of the present invention effectively inhibits both the VEGF and DLL4 signal transduction system.
  • Example 8 Analysis of DLL4 / Notch and VEGF / VEGFR Signaling Pathway Inhibitory Activity of DLL4 / VEGF Double Antibody
  • HUVEC was used in the same manner as used in Example 4 to determine the DLL4 / Notch and VEGF / VEGFR signal transduction inhibitory activity of the double antibody that binds DLL4 and VEGF.
  • the recombinant human DLL4 (rhDLL4, recombinant human DLL4, R & D systems) was diluted with 1 mg / mL of Carbonate buffer solution on 6-well plate (BD), and then added at 1 mL / well, and left overnight at 4 ° C.
  • the control group not treated with rhDLL4 was treated with 1 mL / well of Carbonate buffer solution alone, and left standing at 4 ° C. overnight.
  • HUVECs cultured in 75T plates of passages # 2 to # 5 were taken out during the antibody treatment time, and the cells were removed and cultured as single cells. Wash the cells by centrifugation and resuspend using fresh EGM-2 medium, count the cells and dilute to 5x 10 5 cells / mL, inoculate 1 mL into each well and incubate at 37 ° C, 5%. Incubation was overnight in a CO 2 incubator. EBM-2 minimal medium containing 0.2% FBS was prepared, medium was removed from each well of HUVEC incubated for one day, and once washed with PBS, 2 mL of EBM-2 medium containing 0.2% FBS was treated. .
  • cytolysis buffer solution 1% NP-40, 20mM Tris, 137mM NaCl, 10% Glycerol, 2mM EDTA, 1mM Sodium orthovanabate, 1x Protease & phosphatase inhibitor coctail
  • a cytolysis buffer solution 1% NP-40, 20mM Tris, 137mM NaCl, 10% Glycerol, 2mM EDTA, 1mM Sodium orthovanabate, 1x Protease & phosphatase inhibitor coctail
  • the plate was placed on ice and scraped off the HUVECs of each well with a scraper, collected in a 1.5 mL tube and left on ice. Remove 1.5 mL tubes from the ice every 5 minutes, vortex three times, and then immerse on ice again to proceed with cell lysis, centrifuge (4 ° C, 14000 rpm, 10 minutes) and transfer the supernatant to a new tube. After quantification, the mixture was mixed with 5x SDS sample buffer and boiled at 100 ° C. for 10 minutes, followed by SDS-PAGE analysis.
  • the dual target protein of the present invention can inhibit DLL4 / Notch and VEGF / VEGFR signal transduction pathways as much as the respective antibodies alone.
  • Example 9 Analysis of dual antibody anticancer activity in Avastin-resistant human SCH gastric cancer xenograft model
  • the double-target protein of the present invention significantly increased the anticancer effect against gastric cancer cell lines resistant to Avastin.
  • Avastin (2.5 mg / kg / week) was treated for 3 months to obtain A549 cancer cells resistant to administration of Avastin in which the tumor did not decrease after Avastin treatment. After removing the tumor, Avastin-resistant A549 cells were subjected to Ex-vivo culture and used for the efficacy analysis of the dual target antibody.
  • Avastin resistant A549 lung cancer cell line was inoculated into female nude mice, when the tumor size reached an average of 200 mm 3, each antibody was administered twice a week to in vivo anticancer activity of the dual target antibody. It was confirmed (FIG. 9).
  • In vivo testing using Avastin resistant A549 cells was also performed by administering Avastin-mouse DLL4 surrogate, a double antibody that binds to mouse DLL4 at the same site as human DLL4 epitope, instead of Avastin-DLL4 double antibody targeting human DLL4. Efficacy verification was demonstrated.
  • the double-target protein of the present invention significantly increased the anticancer effect against lung cancer cell lines resistant to Avastin.

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PCT/KR2014/006090 2013-07-09 2014-07-08 Dll4와 vegf에 특이적으로 결합하는 신규 이중표적 단백질 및 이의 용도 WO2015005632A1 (ko)

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ES14823338T ES2742855T3 (es) 2013-07-09 2014-07-08 Nueva proteína de objetivo doble que se une específicamente a DLL4 y CEGF, y uso de la misma
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